Abstract
The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.
Highlights
The error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation
Oxidative damage of DNA is implicated as a cause of aging [1,2,3], carcinogenesis (4 – 6), and a variety of noncancerous diseases such as Alzheimer disease and cardiovascular disease [7] and in the progression to acquired immunodeficiency syndrome in human immunodeficiency virus-infected patients [8]
The radicals nitric oxide (1⁄7NO) and superoxide (O2.) are produced by macrophages and neutrophils upon immune response activation [13, 14]. These radicals combine in a diffusion-limited reaction to form peroxynitrite (ONOOϪ)1 [15], a powerful oxidizing and nitrating agent capable of damaging a variety of biomolecules (16 –18), including DNA [19]
Summary
Oligodeoxynucleotides—DNA synthesis reagents were purchased from Glen Research. Unmodified ODNs were purchased from IDT, Inc. and were purified by PAGE. The purified 19-mer was characterized using MALDI-TOF mass spectrometry (calculated Mr, 5824.8; found Mr, 5825.8) and enzymatic digestion to nucleosides followed by HPLC analysis. The scaffold DNA was removed using the exonuclease activity of T4 DNA polymerase (Amersham Biosciences) by treating with 0.25 units/l for 1 h at 16 °C in a volume of 65 l [36, 37] Under these conditions, scaffold digestion was complete as determined by using a radiolabeled scaffold and analyzing the reaction with PAGE and phosphorimaging. The amount of circular, 19-mer insert-containing genome in each sample was quantified by agarose gel electrophoresis and phosphorimaging. Translesion Bypass Efficiency—An equal amount of each circular genome construct was mixed with 0.025 pmol of internal standard (wild-type circular single-stranded M13mp7L2 DNA) and 100 l of electrocompetent cells. Mutation Type and Frequency—Electrocompetent cells were transformed with each genome construct, as described in the previous sec-
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